Grasas y Aceites

Vol. 53. Fasc. 1 (2002), 45-63

45

Spectroscopy: Developments in Instrumentation and Analysis

By Vincent Baeten and Pierre Dardenne
Quality Department of Agricultural Products. Agricultural Research Centre of Gembloux.
Chausse de Namur, 24. 5030 Gembloux. Belgium.
Tel: 32/81/62 03 50, Fax: 32/81/62 03 88. E-mail: baeten@cragx.fgov.be
CONTENTS
1.
2.

3.

4.
5.

6.

Introduction
Why spectroscopic techniques
2.1. Analytical characteristics
2.2. Spectroscopic characteristics
2.3. Instrumental characteristics
Some theoretical elements
3.1. Classical theory
3.2. Quantum theory
3.3. Absorption and scattering: band position and intensity
Selecting the information
Spectroscopic instrumentation
5.1. Near-infrared instrument
5.1.1. Classical instrument
5.1.2. Microscope and camera
5.1.3. Sample presentation
5.2. Mid-infrared instrument
5.2.1. Microscope and camera
5.2.2. Sample presentation
5.3. Raman instrument
5.3.1. Microscope and camera
5.3.2. Sample presentation
Applications
6.1. Quantitative analysis
6.1.1. Determination of fat content
6.1.2. Determination of major and minor components
in lipid
6.2. Qualitative analysis
References

RESUMEN
Espectroscopa: Desarrollo en instrumentacin y anlisis.
Este artculo de revisin presenta las caractersticas, ventajas, lmites y potencial de tres tcnicas espectroscpicas: las espectroscopias del infrarrojo cercano, del medio infrarrojo y
Raman. Se presentan, y discuten brevemente, los aspectos tericos relacionados con estas tcnicas, la informacin que pueden
suministrar, y las principales caractersticas de la instrumentacin. La ltima parte de la revisin esta dedicada a las aplicaciones de la espectroscopia en anlisis de alimentos, con especial
nfasis en anlisis de lpidos. Las ilustraciones y los ejemplos se
han elegido para demostrar la importancia de las tcnicas espectroscpicas en los procesos en-lnea y en los laboratorios en el
anlisis de componentes mayoritarios y minoritarios.

PALABRAS-CLAVE: Anlisis - Autentificacin Espectroscopia infrarrojo cercano- Espectroscopia infrarrojo medio - Espectroscopia Raman Microscopio Quimiometria.

SUMMARY
Spectroscopy: Developments in instrumentation and
analysis.
This review presents the characteristics, advantages, limits
and potential of three spectroscopic techniques: near-infrared

spectroscopy (NIR), mid-infrared spectroscopy (MIR) and Raman

spectroscopy. The theoretical aspects related with these
techniques, the information that can supplied and the main
features of the instrumentation are presented and briefly
discussed. The last part of the review concerns the application of
the spectroscopy to food analysis, with special emphasis on the
lipid analysis. The illustrations and examples have been chosen to
demonstrate the importance of spectroscopic techniques both in
process (on-line) control and in laboratories for the analysis of
major or minor compounds.

KEY-WORDS: Analysis - Authentication Chemometrics - Midinfrared spectroscopy - Near-infrared spectroscopy - Raman spectroscopy.

1. INTRODUCTION
In recent years, spectroscopic techniques have
come to be regarded as attractive and promising
analytical tools for analyses conducted in research,
control or industrial laboratories. These techniques
are increasingly considered by analysts as an
obvious solution. This trend stems from instrumental
developments, the extensive use of computers and the
development of appropriate chemometric procedures.
Daily, new applications of spectroscopic techniques in
the fields of chemistry, drugs, the agro-food sector, life
sciences and environmental analysis are being
demonstrated and published. The objective should be
not only to extend the field of application of
spectroscopic techniques, but also to use them as a
matter of course in control and industrial
laboratories and to develop (according to
internationally accepted guidelines) fully validated
spectroscopic methods.
This paper presents the characteristics, advantages,
limitations and potential of three spectroscopic
techniques: near-infrared spectroscopy (NIR), mid-infrared
spectroscopy (MIR) and Raman spectroscopy.
Special emphasis is placed on the application of
these techniques to lipid analysis. The illustrations
and examples have been chosen to demonstrate the
importance of infrared (NIR and MIR) and Raman
spectroscopies both in process (on-line) control and
in laboratories for the analysis of major or minor
compounds. There are many books and papers on
the subject; most of the references included here
have been selected for their relevance and clarity.

46

Grasas y Aceites

Table I
Analytical, spectroscopic and instrumental characteristics of NIR, MIR and Raman spectroscopies

INSTRUMENTAL

SPECTROSCOPIC

ANALYTICAL

FEATURES

NIR

MIR

Raman

/
/
-

.
.
.
.
.
-

/
/
-

na
na

na
na

Rapid analysis
Simple analysis: concentration vs peak area
Qualitative and quantitative analysis
None or reduced sample preparation
Standard calibration and transfert
procedures
Validated methods (IUPAC, AOCS, ISO)
Sample form and sample size
No use of pollutant solvents
Economic method
(e.g.
saving of labour
reduction of samples to analyse)
Direct, non-invasive and non destructive
In-situ analysis

z
z

Emission process
Intensity proportional to concentration
W ell-resolved spectra
High resolution spectra
Precise spectral frequency measurement
Non-polar group information
More significant information
Structural selectivity
Signal intensity
Trace element determination
Fluorescence-free spectra
Spectra insensitive to temperature
Sample presentation technique

Push-button instrumentation
W orks well with aqueous samples
Suitable for use at-, on- and in-line process
Compatible with suitable fibre optics
Process monitoring
Microscope
Camera (Multispectral measurement)

2. WHY SPECTROSCOPIC TECHNIQUES

The advantages of NIR, MIR and Raman
spectroscopies are numerous and diversified. Table I
shows the major characteristics of these techniques.
Note that some of these characteristics are the same
as those of other spectroscopic techniques, such as
Nuclear Magnetic Resonance or Ultraviolet. The
characteristics are categorised into three groups:
analytical, spectroscopic and instrumental. For each
characteristic and each technique, a diagram has been
added to indicate whether the characteristic is an
advantage (), a partial advantage ( ) or a
disadvantage (). References in which these
characteristics are discussed are included in the table.

2.1. Analytical characteristics

The analytical characteristics of NIR, MIR and
Raman spectroscopies display certain advantages
that make these techniques attractive as alternatives

/
/
.
/
/
na
/
-

/
.
na
/
.

/
.
-

.
-

/
.
.
.
.

.
/

REFERENCES
(Gerrard 1991)
(Pelletier & Davis 1996)
(Baranska 1987)
(Gerrard 1991)

(Scotter 1997; Murray 1999)

(Gerrard 1991)
(Hendra 1997)

(Vickers &Mann 1991)

(Gerrard 1991)
(Gerrard 1991; W ilson 1991)
(Levin & Lewis 1990)
(Levin & Lewis 1990)
(Terpinski 1987)
(Schrader 1996; Olinga & Siesler, 2000 )
(Olinga &Siesler, 2000)
(Li-Chan 1996)
(Chase 1987)
(Pelletier & Davis 1996)

(van de Voort 1994)

(Vickers & Mann 1991)
(Scotter 1997; Hendra 1997)
(Lewis et al . 1988; Gerrard 1991; Kellerr et al.
al. 1993; Olinga & Siesler, 2000)
19
(Olinga & Siesler, 2000; Ozaki et al. 1992)
(W illiams & Batchelder 1994)

to traditional, tedious and more time-consuming

analytical methods.
First, they are rapid, do not require reagents and
are non-destructive. The best illustration of this is in
protein analysis. The protein content of feedstuffs
and foodstuffs is traditionally determined using the
Kjeldhal method. With this method a single analysis
of one sample takes 3 hours and requires several
millilitres of different pollutant reagents (e.g.,
sulphuric acid) (Bertrand, 2000). Currently, with the
growing concern about analysts safety and
environmental protection, laboratories try to keep at
a minimum the quantity of potentially dangerous
reagents used in their analyses. In addition to the
health and environmental aspects of the use of these
products, it is important to add the cost generated by
the adequate treatment of the chemical waste. An
alternative to the Kjeldahl method is the
determination of protein content by NIR which takes
less than a minute and does not require any

Vol. 53. Fasc. 1 (2002)

reagents. A similar illustration can be provided with

regard to iodine value determination. The time
required for a single determination using a classical
technique (i.e., the titrimetric or chromatographic
method) is 3060 minutes following the procedure
chosen, whereas a spectroscopic determination
takes about 5 minutes. Iodine value can be
measured by NIR, MIR or Raman spectroscopy
(Baeten et al., 2000). For the agro-food sector the
speed of analysis is a crucial factor in process control
where any delay in obtaining the analytical results
may result in significant financial loss. The industry
needs methods that produce rapid results and that
allow eventually to correct the process in progress.
Other analytical characteristics of infrared and
Raman spectroscopies are that they require no (or
limited) sample preparation, that sample form and
size are not a restriction (Williams and Batchelder
1994) and that they can be used for analysing
samples in almost any physical state (i.e., solid,
liquid or gas) and at any macroscopic or microscopic
level. In todays world, where instant answers are
required and nobody has the time available (or to
waste) to prepare samples, there is a need for rapid
methods and simple sampling techniques (Coates
and Sanders 2000).
As discussed before, infrared and Raman
spectroscopies are relatively economic as they are
considerably less demanding in terms of time and
the use of reagents than traditional methods. For
research, spectroscopic methods could be also
economically attractive as they allow the selection
and restriction of number of samples to analyse
using classical techniques. Spectra allow us explore
a population before we determine what we should
measure by classical technique. This is an important
research characteristic of spectroscopic techniques
(Murray 1999). As noted by Ian Murray, Within a
closed population of 1000 samples it is valid to use
spectral selection of a representative set of 100 for
reference analysis, then calibrate and predict the
remaining 900, so saving 90% of the lab effort.
Additional analytical advantages of spectroscopic
techniques are: (i) structural analysis is possible, in
addition to qualitative and quantitative analysis; (ii)
direct, non-invasive and in situ analysis can be
performed. From an analysts point of view, the main
analytical drawbacks of infrared and Raman
spectroscopies are that they are indirect methods,
there is a lack of standardised calibration and
transfer procedures, and there are few fully validated
methods using spectroscopic techniques.
2.2. Spectroscopic characteristics
The spectroscopic characteristics listed in Table I
include
several
spectroscopic
advantages,
particularities and limitations of NIR, MIR and
Raman techniques. Some characteristics are

47

common to all three techniques, whereas others are

specific to only one or two of the techniques. The
characteristics in common are the high resolution of
the spectra obtained and the precise spectral
frequency measurement that facilitates the
mathematical treatment (e.g. spectral subtraction),
as well as the transfer of equation between
instruments. From the spectroscopic point of view, a
general advantage of MIR and Raman
spectroscopies is that bands are well resolved and
can be assigned to specific chemical groups. There
is little ambiguity in spectral assignment and
quantitative analyses can be fairly component
specific (Wilson 1990; Gerard 1991). By contrast,
NIR spectra contain a series of successive
overlapping bands which are difficult to assign to
specific chemical groups. Another characteristic
common to MIR and Raman spectroscopies is that
they have high structural selectivity and contain more
of the type of information needed in structural
elucidation studies (Socrates 1994; Olinga and
Siesler 2000; Schrader 1996). Both techniques can
also provide information on the state of the
components, such as the phase, crystallinity or
conformation (Wilson 1990).
Raman and MIR spectroscopy can be applied
specifically for determining trace elements. One of
the best examples of this is in the measurement of
carotenoid content (tenth of ppm) in food products
using the Raman technique (Li-Chan 1996). The
carotenoid peaks observed in the Raman spectra
are not due to a Raman effect but to a resonance
Raman phenomenon. An example of the MIR
technique coupled with a specific extraction method
is the determination of organochloride at low level in
water (Acha et al. 1998).
A specific advantage of NIR and MIR
spectroscopies is their good signal intensity; in
Raman spectroscopy this is a limitation. Indeed,
Raman scattering is a very weak phenomenon
compared to the absorption observed in the two
other techniques (see section 3). A specific feature of
Raman spectroscopy is that it involves an emission
phenomenon;
Raman
intensity
is
directly
proportional to the concentration of the compound to
be determined and, compared to infrared
techniques, is relatively insensitive to temperature
and works well with aqueous samples (Pelletier and
Davis 1996).
2.3. Instrumental characteristics
One of the most interesting instrumental
advances in spectroscopy is its potential for
developing and commercialising "push-button"
instrumentation. Infrared and Raman spectroscopies
generally appear as sophisticated techniques for
novices in the field and require a trained
spectroscopist to exploit their potential in terms of

48

development work (van de Voort 1994). What is

required is the setting up of dedicated instruments,
including the optimal sample presentation, fully
programmed and precalibrated for target analysis,
such that skilled, specialised operators are not
crucial for the wider use of these techniques. The
development of dedicated instruments in agro-food
analysis has been initiated by the development of
grain and powder NIR analysers and milk MIR
analysers.
Another
instrumental
feature,
making
spectroscopy techniques very attractive for process
control and monitoring, is the possibility of coupling
the instruments with suitable fibre optics. Infrared
and Raman instruments could be applied "at-line" in
the laboratory control, "on-line" with the use of
measurement through optically clear windows and
dedicated sample techniques, and "in-line" with the
use of specific probes for the measurement inside a
pipe or a tank. The field of process analytical
techniques is expanding rapidly due to the economic
benefits achieved by using rapid and accurate
information. As discussed above with regard to
analytical characteristics, this will result in improved
productivity and efficiency, improved quality of the
transformed products and a reduction in cost and in
waste products. The almost instantaneous
acquisition of the analytical results allows a process
to be continuously monitored, and readjusted or
stopped if the desired specification is not achieved.
Currently, NIR spectroscopy is overtaking MIR and
Raman in this field for major technical reasons.
Indeed, if NIR and Raman spectrometry can be used
with relatively low-cost fibre optics of 10100 m in
length without severe signal attenuation, the fact that
Raman measurement focuses on small sample
volumes and has low sensitivity limits its industrial
application. An additional limitation is the safety
argument. Adapted fibre optics based on ZrF 4 and
AgCl are also available for MIR instruments but their
cost, the signal attenuation and their stability restrict
their use to lengths of about 1 or 2 m, which may not
be sufficient for most industrial applications (Olinga
and Sielser 2000; McCreery 2000).
Over
the
past
decade
spectroscopy
measurement has been extended from the
macroscopic to the microscopic domain. Indeed,
several manufacturers now produce NIR, MIR and
Raman microscopes. These instruments have
greatly extended the field of application of infrared
and Raman spectroscopy. They allow the acquisition
of spectra from a small sample area (a few microns)
and are widely used in forensic, pharmaceutical and
artwork analysis. In more recent years we have been
assisting in the development of infrared camera that
allows the simultaneous acquisition of thousands of
spectra of an image. These developments will

Grasas y Aceites

certainly revolutionise the analytical world of

spectroscopy.
3. SOME THEORETICAL ELEMENTS
Generally, the theory of a technique appears
forbidding, and knowledge of the theory is not
essential in the application of that theory. However, a
good understanding of the theory behind a technique
is the best way to improve the quality of its
application in research. The history of spectroscopy
techniques goes back to the work of Frederick
William Herschel in 1800. He discovered that the
suns energy was not limited to what we can see. He
demonstrated this by projecting a rainbow onto a
bench, using a prism. On the bench he positioned a
series of thermometers and measured the relative
heat in different parts of the rainbow. As he moved
from the blue to the red, the heating effect increased
and it continued to increase as he progressed to the
end of the rainbow; moreover, the heating effect
continued well after the end of the rainbow (Davies
1998). Herschels work was the first step in the
discovery of the electromagnetic spectrum.
The electromagnetic spectrum is usually divided
into several regions, including the -ray, X-ray,
ultraviolet (UV), visible (VIS), infrared (IR) divided
in NIR, MIR and far-infrared regions microwaves
and radio wave. Each region corresponds to a
specific kind of atomic or molecular transition
corresponding to different energies. Figure 1
presents part of the electromagnetic spectrum, the
spectroscopic techniques concerned and the
energetic transition involved. The figure also shows
the wavelengths and wavenumbers associated with
each region. Each part of the electromagnetic
spectrum corresponds to one or more spectroscopic
techniques that exploit the phenomenon which
occurs when the electromagnetic radiation of a
determined energy interacts with the molecule. Two
complementary theories have been put forward to
explain the properties of electromagnetic radiation:
the classical theory (electromagnetic radiation is a
wave) and the quantum theory (electromagnetic
radiation is a stream of energetic particles).
3.1. Classical theory
Most of the properties of light can be explained by
radiation with an electric field associated with a
perpendicular magnetic field of high frequency that
moves in the direction of the light. These fields
interact with matter to give rise to a spectrum. The
movement of the radiation has the properties of a
sine wave described by the equation
Y = A sin t

Eq1

Vol. 53. Fasc. 1 (2002)

49

Energy

a)

Overtone and
combination vibrations

Microwave Radio

1st excited
electronic state

Ground
electronic state

b)

Fundamental vibrations

c)

3
2
1

3
2
1

Ultraviolet

hMIR

IR

3
2
1

hNIR

VIS

h
0
h

UV

UV

Frequency

Near-infrared

Mid-infrared

d)

Near-infrared spectroscopy Mid-infrared spectroscopy

(NIR)

(MIR)

800

2500

25000

Ground
electronic state

Wavelength (nm)

Stokes

h0

h0

igh

3
2
1

3
2
1

3
2
1

Raman Stokes

400 50

4000

12500

hanti-

/ q 0

h0

Raman spectroscopy

hRayle

1st excited
electronic state
Virtual state

kes

/q 0

hSto

/ q 0

Rayleigh

Raman anti-Stokes

Wavenumber (cm-1)

Figure 1
Several regions of the electromagnetic spectrum and the related
spectroscopic techniques; the wavelength and wavenumber
limits of each region are shown.

where Y is the displacement with a maximum A

(=amplitude), the angular velocity rad. sec-1 and t is
the time in seconds. In 1 second the pattern will repeat
/2 times, referred to as the frequency expressed
as cycle by second (S -1 or Hz). Equation 1 now
becomes
Y = A sin 2t

Eq2

An additional property of the wave describing the

movement of the radiation is the distance covered in
one complete cycle known as wavelength . The
wavelength is defined by the following equation
= c/

Eq 3

where c is the universal constant of the velocity of

light in vacuum. Traditionally, in NIR absorption bands
are described in terms of wavelengths expressed in
nm. In contrast, MIR and Raman spectra are respectively expressed as the absorbances and the scattering
intensities measured at different wavenumbers
expressed in cm-1. The relationship between wavenumbers and wavelengths (taking into account the
unit used) is (cm -1) = 107/, with the wavelength
expressed in nanometers. Wavenumbers are directly
proportional to frequency by the expression = c
and are easily related to the energy changes involved
in transitions between different vibrational states
(Osborne & Fearn, 1986).
3.2. Quantum theory
The classical theory does not explain all
properties of electromagnetic radiation and
interaction with matter, and fails to account
phenomena associated with the absorption

the
its
for
or

Figure 2
Energy level diagrams (Potential energy versus interatomic
distance) showing the basic transitions involved in NIR, MIR and
Raman spectroscopies. Two electronic states and three
vibrational states are shown.

emission of energy. It is necessary to view

electromagnetic radiation as a stream of discrete
particles. Planck was the first to put forward the
hypothesis that the electromagnetic wave was not
continuous but composed of corpuscular units called
quanta (Osborne and Fearn 1986). The energy of a
quantum of radiation is expressed as follows
E = h

Eq. 4

where E is the energy, the frequency and h the

Planck constant. The quanta of radiation move at a
constant speed and the classical equation c = is
respected but the values of the energy are quantified.
The energy of a quantum of radiation is defined and
characterised by its frequency (Lachenal 1998).
Following this brief outline of the two
complementary theories of the radiation, it is now
appropriate to illustrate the different techniques
discussed in this chapter. The interactions of
electromagnetic radiation with molecules give rise to
several spectroscopic techniques based on
absorption, emission or scattering processes. NIR
and MIR spectroscopies are based on absorption,
while Raman spectroscopy is based on scattering
process. Raman and infrared spectroscopies imply
the discrete vibrational transitions that occur in the
ground electronic state of molecules. It is worth
noting here that a molecule is characterised by its
electronic, vibrational, rotational and translational
energy levels. The vibrational transitions correspond
to various stretching and bending deformation
modes of individual chemical bond. Figure 2
describes the different phenomena observed in the
infrared regions. For the sake of simplification, only
two electronic states (the ground and the first

50

1.2

cis C16:1
trans C16:1
cis C18:1
trans C18:1

0.8

Scatteing intensity

excited) and three excited vibrational states of each

of them are shown in Figure 2.
MIR spectroscopy concerns the region of the
electromagnetic spectrum lying between 4000 and
400 cm-1 (2500-25000 nm). When radiation with
energy corresponding to the MIR range interacts with
a molecule, the energy at defined frequencies can be
partially absorbed. The absorption of the incident
electromagnetic radiation at a particular frequency
(i) is related to specific vibrational excitation energy
(E = hi). The frequencies of these vibrations are
related to molecular parameters such as bending
force constants and masses of the atoms involved in
this vibration. The vibrations under consideration in
MIR are mostly fundamentals (i.e., from the stable
vibrational state to the first excited vibrational state in
the electronic ground state) (Figure 2b).
NIR spectroscopy involves radiation with energy
higher than in MIR (Figure 1). The NIR region lies
between 780 and 2500 nm (12800-4000 cm-1).
Electromagnetic radiation of this region is absorbed
at frequencies corresponding to overtone (i.e.,
corresponding to the transition from the stable
vibrational state to the second [first harmonic] or the
third [second harmonic] excited vibrational state, or a
higher vibrational state), or a combination vibration of
different vibrations of the molecule under
consideration (Figure 2c).
In contrast to the two other techniques, Raman
spectroscopy involves a scattering process. Briefly,
Raman spectroscopy is based on the shift of an
excited incident beam of radiation that results from
inelastic interactions between the photons and the
sample molecules. The region of the electromagnetic
spectrum under consideration is similar to that in MIR
(i.e., 4000-100 cm-1). Figure 2 presents the Raman
effect via an energy level diagram. Let us consider an
incident photon of energy h 0 interacting with a
molecule having vibrational energy levels 1, 2 and
3. In scattering processes, the incident radiation
interacts with the molecule that achieve a virtual (or
temporary) energy level drawn in the figure as dotted
line. The photon of incident energy ho may proceed
with unchanged energy hRay (= ho; i.e., Rayleigh
scattering) or with reduced energy hRST (= ho-h1;
i.e. Raman Stokes scattering) or with increased
energy hRAST (= ho + h1; i.e. Raman anti-Stokes
scattering). According to the Maxwell-Boltzmann law,
only a small portion of the molecules will occupy at
room temperature the first excited vibrational state
(1) of the fundamental electronic state. By
consequence, most of the molecules are in the
ground state, and the Raman Stokes scattering
bands are more intense than the anti-Stokes Raman
scattering bands (Baranska 1987; Diem 1993;
McCreery 2000). Many studies conducted with
Raman spectroscopy involve Stokes Raman
measurement.

Grasas y Aceites

0.6

0.4

0.2

0
1690

1680

1670

1660

Raman shift (cm

1650
-1

1640

1630

Figure 3
FT-Raman spectra of various pure fatty acid methylesters.
Scattered band observed in the vicinity of 1660 cm-1
characteristic of the C=C vibration of RCH=CHR groups.

3.3. Absorption and scattering: band

position and intensity
The so-called selection rules dictate the
probability of excitation of a particular vibration
frequency. The selection rules determine the number
of vibrational bands in infrared absorption spectra
and in Raman scattering spectra. These rules are
derived from the application of group theory to
atomic vibrations of the molecules belonging to
different classes of symmetry. Several factors
determine the vibration frequencies, including: the
inter-atomic distances, the spatial arrangement of
the groups, the Fermi resonance, the physical state
of the sample, the polarity of the environment and
the formation of hydrogen bonds.
Figure 3 illustrates the influence of several factors
on the band position observed in FT-Raman of
different pure fatty acid methylesters (palmitic, C16;
stearic, C18; palmitoleic, cis c16:1; oleic, cis C18:1;
palmitoelaidic, trans C16:1 and elaidic, trans C18:1
acids). The trans fatty isomers have the band of C=C
stretching vibration at a wavenumber higher than the
equivalent of cis isomers (Aparicio and Baeten,
1998).
Infrared absorption requires a change of the
intrinsic dipole moment with the molecular vibration.
In contrast, Raman scattering arises from the
change in polarizability of the electron distribution in
the molecules as it vibrates (Grasseli and Bulkin 1991).
Raman band intensity is proportional to the expression
(/Q)2 where is the polarisability and Q the normal
co-ordinate of the group of atoms of interest. Infrared
band intensity is proportional to the expression (/Q)2
where is the induced dipole moment of the molecule.
This explains why the same molecule may give
infrared and Raman bands with differing intensity
and band shape (Baranska et al. 1987).

Vol. 53. Fasc. 1 (2002)

51

Figure 4
Near-infrared (inverse scale) (a), mid-infrared (b) and Raman
(c) spectra of a vegetable oil. The NIR spectrum is plotted as the
absorbance versus the frequency (nm), Raman spectrum as the
scattering intensity versus Raman frequency (cm-1) and MIR
spectrum as absorbance (Log1/R) versus frequency (cm-1). The
NIR spectrum was obtained by reflection mode while Raman
and MIR (with ATR accessory) spectra using FT instruments.

As in the MIR region, the Raman spectrum of a

vegetable oil (Figure 4c) contains well-defined
bands. Whereas the band position is quite similar for
both regions, the relative intensities of the bands are
very different. For instance, the carbonyl band near
1745 cm-1 is intense in MIR spectroscopy but weak in
Raman spectroscopy. In contrast, the band near
1660 cm-1 of C=C groups is very weak in MIR
spectroscopy but intense in Raman spectroscopy. As
in MIR spectra, Raman spectra also contain
information on the state of the components.
Figure 4a presents the NIR spectrum of a
vegetable oil. NIR spectra show various overlapping
bands. As seen before, these bands are the result of
the first and second overtones, as well as of the
combinations of two or more fundamental vibrations
that occur in the MIR region. From low to high
wavelengths, the region characteristics are: the
second overtones of C-H groups (1050-1250 nm),
the combination band of C-H groups (1300-1450 nm)
and the first overtone of CH=CH (1600-1800 nm),
the OH combination band (1880-1930 nm), and the
CH combination bands (2100-2400 nm).

4. SELECTING THE INFORMATION

5. SPECTROSCOPIC INSTRUMENTATION

Figure 4 presents the (a) NIR, (b) MIR and (c)

Raman (reverse scale) spectra of a vegetable oil. The
attribution of the most noteworthy bands is given. Note
the complementarity of the spectroscopic techniques
from the chemical point of view. A brief description of
the band is given here. For a more detailed description
of the different spectra, and of the attribution of the
bands in fats and oils, readers are referred to the
literature (Socrates 1994; Baeten et al. 2000).
The MIR spectrum (Figure 4b) of a vegetable oil
or fat contains well-defined peaks, mainly in the
3100-1700 cm-1 region, and overlapping peaks in the
fingerprint region between 1500 and 700 cm-1. As
described earlier, this region of the electromagnetic
spectrum contains mainly fundamental vibration
bands when the vibration induces a change in the
dipole moment of the molecule under consideration.
From high to low wavenumber, we have at around
3000-2800 cm-1 a group of peaks of C-H stretching
vibration, near 1745 cm-1 a single peak being the
C=O stretching vibration of carbonyl groups, in the
1400-1200 cm-1 region the bending vibration band of
CH2 and CH3 groups, and in the 1125-1095 cm-1
region the bands characteristic of stretching vibration
of C-O and C-C groups. In addition to this
information, MIR spectra can also provide
information on the state of the components such as
the phase, crystallinity and conformation (Wilson
1990). The main spurious bands observed in MIR
spectra are those of CO2 and water vapour. For
tables regrouping the band position of the different
groups, readers are referred to Socrates (1994).

The field of instrumentation in infrared and

Raman spectroscopies is constantly evolving. This
evolution has been rapid, for instance, in process
analytical chemistry which is critical for the
pharmaceutical, chemical and agro-food industries,
among others. A general trend is that the analysis is
moving closer to the sampling point by means of fibre
optics allowing real time analysis and continuous
control of processes. Another trend is the
development of analytical chemistry systems
combining the instrument, the interface between the
instrument and the sample (i.e., sample presentation
technique) and the software integrating data
acquisition, chemometrics and data archiving for
specific application (Ciurczak 1991). These systems
are usually called dedicated instruments.
In addition, with the introduction of hyphenated
techniques such as infrared microscopy, the
application of spectroscopic techniques extends
from macroscopic analysis to microscopic analysis.
These and other developments enable infrared and
Raman spectroscopy to be applied to more and
more analytical challenges. In the following sections,
the main instrumental characteristics are described
for each of the techniques covered in this chapter. As
mentioned earlier, special emphasis has been
placed on the instrumentation used (or that could be
used) in fat and oil analyses.

1300

1500

1700

1900

Fingeerprint region
C-C, C-O bending
trans -CH=CH- bending

-CH2, -CH3, C-H bending

-COO-, C=O stretching

b)

-CH=CHC-H Combination

-CH2,-CH3, -CH=CHC-H 1st overtone

-OH
Combination

-CH2,-CH3
C-H 2nd overtone
1100

-CH2,-CH3, H2O
Combination, O-H 1st overtone

NIR

c)
-CH2, -CH3
C-H stretching

-CH2, -CH3
C-H Combination

a)

-CH=CH-, C=C stretching

cis -CH=CH-, C-H stretching

Raman

MIR
2100

Wavelength (nm)

2300

2500

3100

2700

2300

1900

1500

1100

700

Wavenumber (cm-1)

5.1. Near-infrared instrument

Various classifications exist to describe the NIR
spectrometers. One of them classifies the

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instruments into three groups according to the

instrumental device: (i) sequential instruments,
where the absorbances are collected sequentially in
the time (in this group are the instruments equipped
with a monochromator or filters); (ii) multiplex
instruments (FT), where the detector simultaneously
receives information from several frequencies; and
(ii) multichannel instruments, with several detectors
that separately record the absorbances at several
wavelengths. Another classification takes into
account the dispersive or non-dispersive properties
of the instrument. With a dispersive instrument, the
frequencies are spatially separated by means of an
appropriate device such as a prism or a
monochromator. In contrast, the main feature of
non-dispersive instrument is the use of
interferometry technology (Bertrand 1998).
5.1.1.

a) Transmission mode
Source

Wavelength
selector

Sample

Detector

Polychromatic radiation

NIR spectrum

b) Reflection mode

Wavelength (nm)

Detector
Source
Polychromatic radiation

Wavelength
selector

Sample

Detector

Figure 5
Schematic configuration of a dispersive NIR instrument;
(a) transmission mode and (b) reflection mode.

Classical instrument

Most of the instruments used in agro-food

analysis are sequential instruments equipped with a
monochromator or a filter wheel as a wavelength
selector. Figure 5 shows the instrumental
configuration of these instruments working in
transmission or reflection mode. Briefly, a
spectrophotometer includes a polychromatic
radiation source (e.g., a tungsten-halogen lamp), a
wavelength selector, a sample compartment and the
detector. The wavelength selector could be a
monochromator, a filter wheel, an acoustico-optic
tuneable filter (AOTF) or an interferometer. A
monochromator disperses the radiation according to
wavelength. Holographic grating is the most popular
dispersion element used. The filter wheel is a device
allowing the sequential bands determined by the
interference filter. The wheel usually holds between 3
and 20 filters. The AOTF technology is based on a
monocrystal of Tellurium dioxide (TeO 2) coupled with
a thin piezo-electric monocrystal plate. AOTF is
based on the modification of the refraction index of
matter by means of radio frequency effect. When a
high-frequency electric field is applied, the piezo
plate generates a high-frequency acoustic wave
propagating in the crystal. If NIR radiation is beamed
simultaneously with the acoustic wave onto the face
of the crystal, both will interact and part (i.e., band
with a determined wavelength) of the incident
radiation will be refracted. With a change in the high
electric field applied to the piezo-plate, the band with
the position of the transmitted light is shifted. Other
dispersive instrumental devices include the use of
several light emitted diodes (LED) as a light source in
the spectrometer. With such a system each LED is
successively activated and the respective
absorbances measured. The instruments based on
AOTF or LED technology have the advantage of not
including moving parts and are quite robust for
industrial application.

In addition to the instruments described above,

there is multichannel detection using diode array
technology. With such an instrument, several
detectors are used and can record simultaneously
the reflected light. For example, a Corona-Zeiss
instrument adapted on an experimental hopper
developed and designed at CRAGx to simulate the
flux of a harvester, or a Perten instrument used to
measure fruit and meat products. These instruments
are used at CRAGx to measure the quality of grain or
forage in the field (Sinnaeve et al. 2000) and to check
the quality of fruit (Moons and Dardenne 2000) as
well as the authenticity of farmed chicken (Fumire
et al. 2000). In the first study the instrument is placed
on the harvest engine, while in the two others the
fruit or the entire chicken is placed below the
instrument. Concurrently, there is also a trend
towards the miniaturisation of the instrument.
5.1.2.

Microscope and camera

All the instruments presented hitherto are used to

analyse large volumes of samples with a macro
sampling configuration. In the past 20 years,
spectroscopic techniques have been used in the
analysis of much smaller micron-sized samples
using optical microscopes interfaced with existing
spectrometers (Williams and Batchelder 1994). In
forensic analysis, infrared microscopy has become a
standard laboratory procedure. An important factor is
the ability to record visual images of the sample and
to link these to the infrared signature of the sample at
a specific location. However, it is important to
remember that infrared and Raman microscopies
are still microscopic and by nature require a degree
of expertise for good sample preparation and data
acquisition procedures (Coates and Sanders 2000).
Several companies active in NIR instrumentation
produce NIR microscopes. These instruments allow
spectra to be collected from extremely small samples

Vol. 53. Fasc. 1 (2002)

53

a) FT-NIR microscope

c) Chocolate spectrum
Polychromatic radiation

1 ,4

Source

Interferometer

1 ,3

A bs o r ba nc e ( Lo g 1 /R

1 ,2

1 ,1

Fixed miror

0 ,9

0 ,8

0 ,7

0 ,6

0 ,5
1 1 00

1 30 0

1 50 0

1 7 00

1 90 0

2 1 00

2 3 00

2 50 0

W a v e le ng t h ( n m )

litt
sp
am
Be

er

d) Hazelnut spectrum
Moved miror

b) Bar of Belgian chocolate

MIR spectrum

1, 1

a b s o rb a n c e ( L o g 1 / R

0, 9

Sample

0, 8

0, 7

Fourier
transform

0, 6

0, 5
11 00

1300

150 0

1 700

1900

21 00

2 300

250 0

W a v e le n g t h ( n m )

Wavenumber (cm-1)

Detector
Figure 6
Perkin-Elmer FT-NIR microscope (a) and Visible image of the
cross-section of a bar of Belgian chocolate with hazelnut
pieces (b). Spectra of chocolate (c) and the hazelnut (d).

(5 m x 5 m). Usually, the instrument includes a

camera and a viewing system for magnifying the
visible light image of the sample so as to observe,
identify and isolate a point of interest. For example,
Perkin-Elmer FT-NIR microscope (Figure 6) is
used at CRAGx to detect meat and bone meal
particles in feedstuffs (Piraux and Dardenne
1999).
The infrared microscope can be used to take
spectra from a variety of sample points from an
inhomogeneous surface (for instance, a variety of
flour particles) and produce an infrared map. The
procedure used is called mapping and is very time
consuming because the measurement has to be
done point by point. In recent years, the introduction
of the multichannel spectrometer has been made
more efficient and allowed the simultaneous
acquisition of spectral data from several points. With
multichannel instruments, several photoelectric
detectors are used and allow a simultaneous
recording of the reflected or transmitted light
corresponding to several points. Basically, there are
systems involving a line-scan camera or a matricial
camera (two-dimensional detector).
5.1.3.

Sample presentation

The presentation techniques used in NIR

spectroscopy are numerous and depend on the
transmission and reflection mode or the state of the
sample analysed (gas, solid or liquid). For liquids
such as oils, special reflection and transflexion cells
are designed for the reflection mode. For the
transmission mode, there are specially designed
quartz cuvettes or flow cells. These devices are very
attractive for analysing oils and fats as they can be
thermostatised. Usually, these cells have a path
length of 0.5 or 1 mm. A greater path length would
lead to saturation of the detector except in the near

Figure 7
Schematic configuration of an FT-MIR instrument.

near-infrared (800-1100) where the path length can

reach 2-3 cm. For the reflection mode and for solid fat
samples such as butter and margarine, there is
aluminium cup covered with a quartz window.
5.2. Mid-infrared instrument
The first generation of MIR spectrometers used a
high-resolution diffraction monochromator and has
been used mainly for qualitative analysis such as the
identification of chemical compounds. The
quantitative application of these instruments has
been limited. In early 1970 the MIR instruments
underwent an important revolution with the
development of commercial spectrometers using
interferometry technology. This revolution sparked a
renewed interest in this technique (Wilson and Tapp
1999). Figure 7 presents the schematic diagram of
an FT-MIR instrument.
An interferometer consists of two perpendicular
mirrors, one is stationary, while the other moves at a
constant velocity. Between these mirrors there is a
semi-reflector (also called beam-splitter). The beam
enters the interferometer, hits the beam-splitter and
is divided into two beams. One beam is reflected
onto the fixed mirror and the other onto the moving
mirror where they are reflected to be recombined at
the beam-splitter. The two beams undergo
constructive and destructive interferences due to the
path difference between the two mirrors (Griffiths
1972; van de Voort 1994; Diem 1993). The
recombined radiation is directed to the sampling
compartment where it will interact with the sample.
The transmitted, diffused or reflected light is pointed
at the detector where fluctuations in the intensity of
the energy are digitised, resulting in an interferogram
that contains the spectral information related to the
sample. The interferogram is the intensity of energy
measured versus the position of the moving mirror

54

Grasas y Aceites

(time domain) and is converted into a conventional

infrared spectrum (frequency domain) by the
mathematical operation known as the Fourier
transformation.
Thus,
spectroscopy
using
interferometry is also called Fourier transform (FT)
spectroscopy.
There are several advantages of interferometry
technology. First, interferometers have a higher
spectral optical conductance (or optical efficiency)
than the grating spectrometers (i.e., instrument using
the monochromator technology). This characteristic
is called the Jacquinots advantage. Second, there is
the Felgetts advantage (also called the multiplex
advantage), which emphasises the efficiency of the
interferometer in using the light entering it. The third
advantage is that the frequency scale is generated
from a He-Ne laser, the wavelength of which does
not vary and is known precisely. This enables spectra
to be superimposed exactly and added. This is called
the Connes advantage (Wilson, 1990; Chase, 1987;
Hendra et al., 1993).
5.2.1.

Microscope and camera

As discussed in the NIR instrumentation section,

joining light microscopy with infrared spectroscopy
allows scientists to resolve the chemistry of
microscopic domain. In 1980, the commercial MIR
microscope became available. By late 1980, 4000
FT-MIR microscopes were in use in a variety of fields,
including the analysis of forensic trace evidence, the
detection of microscopic contamination in drugs, and
the analysis of defects in industrial products (e.g.,
fibre coatings) (Wetzel and Reffner 2000). In the past
few years MIR cameras have come onto the market,
but their application in agro-food analysis has not
been investigated.
5.2.2.

Sample presentation

Concurrently with the development of FT

instruments, the development of adequate sampling
presentation techniques for the MIR region has
contributed to the renewed interest. Indeed, the
improved performance of FT-MIR instruments made
it possible to design sampling accessories that were
difficult to implement using older instruments. Initially,
the sample presentation techniques included fixed
path length transmission cells, coated or smeared
films on windows, hot-pressed films and alkali halide
pellets (KBR) were used.
Now there are more user-friendly sample
presentation techniques for MIR. Possibly the most
interesting development is the introduction of simple
reflectance techniques that include specular
reflectance for solids (e.g., powder), attenuated total
reflectance (ATR) for liquid or slurries. As this chapter
focuses on the analysis fat and oils, the ATR
technique is outlined here as it is aimed at liquids and

a)

b)

c)

Figure 8
Several sampling devices used in MIR spectroscopy. a) ATR,
b) Thermostatised flow cell, c) IR-card.

slurries. In ATR, the sample with a refractive index is

in contact with a crystal (also called internal
reflection element [IRE]) with a higher refraction
index and with a weak infrared absorption in the
region of interest. The radiations are combined in the
IRE and reflected on its inner surface. At each
reflection, a standing evanescent wave exists and
part of the radiation energy penetrates a short
distance into the sample and interacts with it. The
penetration depth of the radiation depends on the
incidence angle, the refractive index of both
materials (crystal and sample) and the wavelength
(Harrick 1967). Several varieties of ATR exist:
cylindrical ATR, squarecol ATR and horizontal ATR.
The first two have an effective surface several times
greater than the third (Harrick 1967; Afran 1993). For
fats and oils we have thermostatised flow cells and
ATR cells that could be coupled with an appropriate
pump. Other sampling accessories sucha IR-card in
polyethylene can be used with success in analysis of
fats and oils. Figure 8 shows different sampling
techniques that can be used in MIR spectroscopy.
5.3. Raman instrument
In the past two decades, Raman instrumentation
has changed a lot. Indeed, with the introduction in
1986 of the near-Fourier transform (FT) Raman
spectrometer including the charged coupled device,
the use of diode lasers emitting in the NIR range and
holographic optics have led to a resurgence of
interest in this technique in agro-food analysis
(Chase 1987). There are two fundamentally different
designs of the Raman spectrometer: dispersive or
non-dispersive. There are many problems
associated with dispersive instruments, such as
fluorescence interference, photodecomposition of
the sample, wavenumber calibration problems, lack
of precise frequency base from scan to scan, and
difficulty in attaining high-resolution spectra (Chase

Vol. 53. Fasc. 1 (2002)

55

Interferometer
Fixed miror

Laser

Sample
Scattering radiation

Monochromatic radiation

li
sp
am
Be

r
tte

Moved miror

Scattering intensity

Detector
Raman spectrum

Raman shift (cm

-1

Fourier
transform

Figure 9
Schematic configuration of an FT-Raman instrument.

1987). The main difficulties in the fats and oils

analysis
with
classical
dispersive
Raman
spectrometers are caused by the fluorescence due
to sample impurities, specifically the carotenoid
content (Levin and Lewis, 1990; Sadeghi-Jorabchi et
al. 1990). As the non-dispersive spectrometer seems
better with regard to the analysis of fat and oils, only
this instrument will be discussed here (Diem 1993;
McCreery 2000). Figure 9 presents a schematic
non-dispersive NIR FT-Raman spectrometer.
In a near-FT-Raman spectrometer, a laser
(monochromatic beam emitting in the NIR range) is
directed with mirrors onto the sample. The incident
photons interact with the samples and scattered light
is collected by adapted optical devices and directed
to the interferometer. The modulated beam reaches
the detector and the Raman spectrum is obtained by
the application of Fourier transformation on the
signal (i.e., interferogram) measured at the detector.
Note that in an FT-Raman instrument the
interferometer is placed after the sample, while in an
FT-MIR instrument it is located before the sample.
The advantages of interferometry discussed earlier
in relation to MIR instruments are also valid for
FT-Raman instruments.
5.3.1.

Microscope and camera

As in the case of NIR and MIR spectroscopy,

Raman spectroscopy can be used for the analysis of
very small samples or small heterogeneities in larger
samples. This potential was first demonstrated in
1975 by Rosco and colleagues (ref. cited by Williams
and Batchelder 1994). In fact, the ability of Raman
spectroscopy to utilise conventional glass optics
made it possible to interface a classical Raman
spectrometer with an optical microscope. In
comparison with the instruments discussed earlier,
the Raman spectrometer used in Raman microscopy
generally has an emitting source in the visible range.

Current Raman microscopes are able to analyse

samples down to 1 m in diameter, making this
technique very attractive for micron-sized studies.
The ability of this technique to obtain the vibrational
fingerprint of fundamental vibrations from a
micro-scale sample or from an impurity in a large
matrix is also a decisive factor in its application in
research and industrial laboratories (Williams and
Batchelder 1994). The examples of applications of
Raman microscopy are numerous. For instance, it is
used in the analysis of small impurities in
pharmaceutical tablets, polymer film, minerals,
human dentin and resin composites, carbon fibre
and micro-organisms (McCreery 2000). In addition,
the observation of very weak water bands in Raman
spectroscopy enables the recording of spectra from
aqueous phase media and permits the application of
Raman microscopy to the analysis of in situ cells
such as human tissue (McCreery 2000) or vegetable
products (Manfait et al. 1999).
Raman microscopes can be used to produce a
Raman map of a sample by the successive acquisition
of the spectra from several points. This process of
mapping has been made more efficient by using 2D
detectors and collecting line images or global images.
Raman line and global imaging have been used for the
study of products as different as semiconductors or
human lymphocyte (McCreery 2000).
5.3.2.

Sample presentation

Raman spectroscopy in chemical analysis

combines many of the advantages of MIR and NIR
spectroscopy. As discussed earlier, MIR and Raman
spectroscopies concern the fundamental vibrations
with high spectral resolution. On the other hand,
sampling in NIR and Raman spectroscopies can be
very simple (Bailey et al. 1967). The Raman spectra
of samples contained in glass (NMR tube or trial
tube) or plastic vials can be easily obtained without
sample preparation. Care needs to be taken for the
variation (e.g., in thickness) between sample
containers. Also, spectra from very small quantities
(as small as 1 l of solution or 1 mg of solid sample)
can be obtained (LiChan, 1996). As for NIR, low-cost
optical fibres can be used.
The development of NIR, MIR and Raman
instrument is closely linked to the calculation
capacity of todays computers, as well as to the
widespread use of mathematical and statistical
procedures. In fact, according to the complexity of
the structural attribution of absorption and scattered
bands, as well as the high quantity of data rapidly
generated by spectroscopic techniques (usually
several hundreds or thousands of data with high
correlations obtained in a few seconds or minutes),
mathematics and statistics are unavoidable (Martens
and Naes 1989; Williams and Norris 1987). The
chemometrics term regroups the mathematical and

56

Grasas y Aceites

6. APPLICATIONS
Various books and papers have reviewed the
application of near-infrared (NIR) (Baeten et al. 2000;
Riaublanc et al. 2000), mid-infrared (MIR) (Wilson
1990; van de Voort and Ismail 1991; van de Voort
1994; Wilson and Tapp, 1999; Baeten et al., 2000;
Riaublanc et al. 2000; Bertrand and Dufour 2000)
and Raman spectroscopies (Ellis et al. 1989; Li-Chan
1996; Keller et al. 1996; Baeten et al. 2000) in the
analysis of agro-food and lipids. The following
sections summarise the potential and the trends in
lipid analysis (quantitative, qualitative, process
monitoring).

and public networks of NIR instruments for analysing

cereals, forage or other agro-food products. In this
structure, a mathematical model is developed in one
laboratory and has to be transferred to the different
partners of the network (Dardenne and Biston 1991;
Bouveresse et al. 1994; Dardenne and Welle 1998;
Duponchel et al. 1999). The Agricultural Centre of
Gembloux is charged with the management of this
network by Provimi Research and Technology
Centre. The network includes 12 feed factories
equipped with NIR spectrometers and spread out
across the world. The advantage of a network is the
saving of time and cost by the reduction of samples
to analyse using the classical technique, as well as
the acquisition of very robust calibrations (regression
models). Among the limitations of such a structure
are the need to standardise the different instruments
of the network to facilitate the smooth transfer of data
between them and the need to standardise the
references laboratories of the network.
The ultimate evolution of the network is the use of
Internet facilities to manage it. In a classical network,
the different spectrometers are standardised and
hold the global equations of the network to predict
unknown samples. In a network using Internet
facilities, the spectrometers are also standardised,
but the equations are stored by a common server. To
predict an unknown sample, the spectrum is sent via
the Internet to a server were the equations are
stored. There, the determination of the selected
component is done and the result is sent by
electronic mail to the laboratory. The great advantage
of such a procedure is the ease of managing the
different equations of the network because updates
of equations need to be done only on the server and
not on each spectrometer, as in the case of a
classical network. An additional advantage is the
cis =C-H

statistical approaches that are especially suitable for

handling the large datasets produced by modern
analytical methods such as infrared and Raman
spectroscopies. Such data contain a large number of
discrete points. In general, there are more data
points per observation than there are observations
(Kemsley 1996; Fearn 2001). Today, the research
activities in chemometrics are very intense and a
significant proportion of publications dealing with
spectroscopy focus on this field.
In infrared and Raman spectroscopy, the
analytical problems addressed by chemometrics
tend to fall into two classes: qualitative analysis and
quantitative analysis. Although some of the
chemometric techniques used in spectroscopy
overlap the two classes partially or totally, each of
them corresponds to two main varieties of
algorithms: discriminant algorithms for the qualitative
analysis and multiple regression algorithms for
quantitative analysis (Howard 1989; Brown 1995;
Aparicio 2000; Dardenne et al. 2000). As there is a
special chapter on chemometrics in the book, this
topic unavoidable with spectroscopic methods is
not developed.

6.1.1.

Determination of fat content

Infrared techniques are widely used to determine

the main constituents of agro-food and feed
products. For instance, NIR spectroscopy can be
applied to determine the fat content of agricultural
products such as cereals, foodstuffs such as butter,
milk, flour or meat products, and feedstuffs such as
fish meal or pet food (Sinnaeve et al. 1990; Chevalier
et al. 1990; De Pedro et al. 1992; Panford and de Man
1990; Garcia-Olmo et al. 1998; Laporte and Paquin
1999). As discussed earlier, this technique can be
used to determine the constituents of a wealth of
varieties of samples, whether liquid or solid, ground
or whole.
One of the trends is the development of networks
of infrared instruments. Worldwide, there are private

trans =C-H

6.1. Quantitative analysis

Figure 10
Raman scattering bands observed in the vicinity of
1630-1690 cm-1. Spectra of milk anhydrous fat having a trans
content between 0.5 and 12%.

Vol. 53. Fasc. 1 (2002)

high flexibility of such a procedure where a

laboratory that has to analyse an unusual product
can check the server to see whether the equations
for this product had been developed for another
laboratory of the network. The main drawback of the
use of the Internet is the need of permanent
connection. But batch mode can be used.
Mid-infrared spectroscopy is also widely used to
determine the fat content of high fat products such as
butter and margarine or milk products. The dairy
industry uses MIR spectrometry in routine analysis
and the official control laboratories make the analysis
with this kind of instrument (Wilson 1990; van de
Voort et al. 1992, 1993).
6.1.2.

Determination of major and minor

components in lipid

The demonstration of the potential of IR and

Raman spectroscopies for the determination of
various chemical compounds and chemical indexes
have been done. Briefly, the different determinations
are: (i) the unsaturation degree known as iodine
value (NIR: Hourant et al. 2000; MIR: Afran and
Newbery 1991; van de Voort et al. 1992b; Sedman et
al. 2000; Raman: Sadeghi-Jorabchi 1990; Baeten et
al. 1998); (ii) the trans and cis content (NIR: Li et al.,
2000; MIR: Belton et al., 1988; Ulberth and Haider
1992; Sedman et al. 1997; van de Voort et al. 1995;
Mossoba et al. 1996; Degreyt et al. 1998; Adam et al.
1998, 1999; Favier et al., 1996; Alonso et al. 2000;
Raman: Bailey and Horvat 1972; Sadeghi-Jorabchi
et al. 1991); (iii) the saponification number (MIR: van
de Voort et al. 1992; Li et al. 2000); (iv) the solid fat
index (MIR: van de Voort et al. 1996); (v) the free fatty
acids (MIR: Lanser et al., 1991; Ismail et al. 1993);
(vi) the peroxide and anisidine values (MIR: van de
Voort et al. 1994; Dubois et al. 1996; Ma et al. 1997,
2000). Near-infrared spectroscopy is also used to
analyse the fatty acid profile of high-fat products
Alonso et al. 2000). Figure 10 shows the Raman
scattering bands observed in the 1630-1690 cm-1
region of milk anhydrous fat having a trans content
between 0.5 and 12% (Baeten, V., Meurens, M.;
Larondelle, Y. and Dardenne, P., Unpublished
results). Today the trend lies in the development of
validated spectroscopic methods based on
internationally
accepted
guidelines.
Various
collaborative studies of spectroscopic methods have
been published (Adam et al. 2000; Cox et al. 2000).
Fulfilling certain experimental and sampling
criteria, spectroscopic techniques can be used to
analyse minor compounds. This is particularly true
for Raman spectroscopy, although the development
of extractive methods for MIR facilitates the detection
of compounds at a very low level (Acha et al. 1998;
Yang and Huan 2000). The sensitivity of a
conventional Raman analysis is usually at the
percent level. However, by creating certain response

57

conditions with the laser wavelength used to

illuminate the sample, analytical sensitivities can be
determined at the ppm and pbb levels (Williams and
Batchelder 1994; Keller et al. 1993; Schrader 1996).
These sensitivities are due to the Raman resonance
phenomenon and not to the classical Raman
phenomenon that occurs in Raman experiments.
Resonance Raman spectroscopy has been applied
to analyse selectively trace components such as
pigments (Merlin et al., 1994; Ozaki et al., 1992),
cholesterol (LeCacheux et al. 1996) and vitamin A.
Another way to obtain an enhanced Raman signal is
to use surface-enhanced Raman spectroscopy
(SERS) (Dou et al. 1999). In SERS, the analysed
molecules are adsorbed onto a microscopically
roughened metal surface and the Raman
measurement is then made. Surface enhancement
spectroscopy has been applied successfully to lipids
(Saint-Pierre Chazalet 1994; Weldon et al. 1998).
6.2. Qualitative analysis
The role of the analyst in the agro-food sector is
evolving rapidly. One trend is the shift of interest from
compositional legislation to label declaration. Thus,
the authentication of agro-food products has become
a crucial issue over the past decade (Simpkins and
Harrison 1985; Dennis and Ashurst 1996).
Authentication is usually defined as the assessment
of a product to ensure that it conforms strictly with
the description provided by its label and complies
with the legislation in force in the country in which it
is marketed or sold (Martin 1996; Aparicio et al.
1996; Lees 1998; Aparicio 2000). Authentication is of
paramount importance both for official bodies in
charge of the control of labelling and for the industry
where incoming batches of raw materials and
finished products must be tested for compliance with
specifications. Spectroscopic techniques are widely
used to tackle authentication challenges. The fact is
that infrared and Raman spectroscopy can provide in
a few minutes spectral signatures that can be used
as a fingerprint of the agro-food products. Table II
summarises some of the papers showing the
potential of NIR, MIR and Raman spectroscopy in
authentication. The table is not exhaustive, but
presents the various agro-food products analysed,
the main issues and the spectroscopic method used
in the references listed.
Different features can be extracted from the
references listed in Table II. First, the studies can be
classified according to whether real or artificial
samples were used. Second, two strategies to
demonstrate the potential of the techniques were
used. (i) Some of the works are based on the
analysis of a sample set (usually several tenths) of
authentic and adulterated samples and use different
chemometric tools to demonstrate the potential of
spectroscopic techniques. (ii) In contrast, other

58

Grasas y Aceites

Table II
Review of papers on the authentication of food products using infrared (NIR, MIR) and Raman
spectroscopy. (Modified from Baeten & Aparicio 2000)
Food product

Issue

Analytical
method

Cereals

Rice grains

Geographic origin

NIR

Coffee

Whole dried or/and ground

coffee beans

Species
discrimination

NIR
FT-MIR

NIR
FT-MIR

Adulteration
Dairy
products

Milks
Butters
Skimmed milk powder

Adulteration
Adulteration

NIR &
FT-MIR
NIR
NIR

Fruit-based
products

Fruit juices

Geographical origin
and species
discrimination

NIR

Fruit pures
Jams

Meat

Meats

Adulteration
Fruit concentration
Fruit type
Process
discrimination

Oils and fats Edible oils and fats

fats

FT-MIR
FT-MIR
FT-MIR

Defernez & Wilson 1995

NIR

Downey & Beauchene

1997a
Downey & Beauchene
1997b
Thyholt & Isakson 1997
Thyholt et al. 1997
Thyholt et al. 1998
Bewig et al. 1994
Safar et al. 1994
Sato 1994
Hourant et al., 2000
Dupuy et al. 1995b
Dupuy et al. 1996
Lai et al. 1994
Guillen & Cabo 1997
Marigheto et al. 1998
Baeten et al. 1998a
Baeten et al. 1998b
Marigheto et al. 1998
Baeten & Aparicio 2000
Wesley et al. 1995
Wesley et al. 1996
Lai et al. 1995
Li-Chan 1994
Baeten et al. 1996
Aparicio & Baeten 1998
Davies et al. 2000
Bertran et al. 2000

NIR

FT-MIR

Raman

Virgin olive oil

NIR
Adulteration
FT-MIR
Raman

NIR
Geographic origin

studies try to calibrate the spectrometer to detect

compounds known as indicators in the detection of
food product adulteration. These strategies are
complementary and need to be investigated when a
study is done on the potential of a spectroscopic
technique (Aparicio and Baeten 1998). One example
is the successful attempt to calibrate the Raman or MIR
spectrometers for the measurement of trilinolein (LLL)
in virgin olive (Baeten et al. 1996; Aparicio and Baeten
1998). In this study, virgin olive oil spiked with LLL was

Osborne et al. 1993

Krzanowski 1995
Downey et al. 1994
Downey & Boussion 1996
Dupuy et al. 1995a
Kemsley et al. 1995
Briandet et al. 1996a
Downey & Spengler 1996
Briandet et al. 1996a
Briandet et al. 1996b
Collomb & Spahni 1991
Sato et al. 1990
Downey et al. 1990
Downey et al. 1992
Scotter & Legrand 1994
Twomey et al. 1995
Li et al. 1996
Kemsley et al. 1996
Defernez et al. 1995

NIR
Species
discrimination
Adulteration
Species
discrimination

Reference

used to calibrate the spectrometer. Trilinolein

(determined by liquid or gas chromatography) is a
chemical indicator which allows the detection of
adulteration of virgin olive oil with seed oils to be traced
(Aparicio 2000). Similarly, in a recent study it was
demonstrated that an NIR spectrometer could be
calibrated to quantify different chemical parameters
(e.g., protein, water and fat content) and then to use the
results to check the samples compliance with the
specifications for Belgian hams, Jambon dArdenne

Vol. 53. Fasc. 1 (2002)

(Dardenne et al., unpublished results). The

chemometric technique used in the papers listed in
Table II involves two procedures: (i) working with a
determined number of variables (i.e., absorbances or
scattering intensity) or (ii) working with all the
variables using condensed chemometric techniques
such those based on principal component analysis.
In the past few years, an additional chemometric
procedure for authentication with spectroscopic data
has been proposed by various authors; it is based on
hierarchical classification (also called sequential
stepwise identification) to address authentication
problems (Aparicio and Baeten 1998; Baeten and
Aparicio 2000). With this procedure, a series of
classification rules are constructed (according to the
same or different multivariate techniques) and
organised to authenticate a food product. The
hierarchical procedure is very attractive as it allows
for instance the combination of different discriminant
equations, with data coming from different spectral
regions or different spectroscopic techniques.
Although the papers listed in Table II demonstrate
the potential of NIR, MIR and Raman spectroscopy
in the authentication of food products, none
addresses the feasibility of using these methods in
the daily control of authenticity. To achieve this goal, it
will be necessary to develop the tools for the
construction, management and exploitation of the
reference databases. Indeed, all the developed and
proposed methods based on spectroscopy require a
relevant references database. The appropriate
chemometric tools for exploiting this database need
to be developed and standardised.
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